Scaffold for Soft Tissue Augmentation and Reinforcement

All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety.

This application claims priority from AU2021902826 filed 31 Aug. 2021 and AU2022900504 filed 3 Mar. 2022, the entire contents of which are herein incorporated by reference.

The disclosure relates to a scaffold graft material, and method of production thereof, for tissue repair and/or support (e.g., reinforcement). In one particular application, the scaffold material is used for treating pelvic organ prolapse (POP).

Pelvic organ prolapse (POP), including vaginal prolapse is a common condition in women with an incidence of 40-60% and a lifetime risk of surgery of 12-19%. In Australia, for example, it has been estimated that over 50% of women who have given birth vaginally are at risk of POP to some degree (www.contienence.org.au). However, childbirth is not the only cause of POP in women (e.g., gynaecological cancer treatment and heavy lifting, age and obesity are also common causes; Ramaseshan A S et al.,29(4):459-476, 2018), so that the actual incidence of POP among Australian women can be expected to be significantly higher.

POP is characterised by a descent of the pelvic organs (e.g., vagina, bladder, uterus and bowel) from their normal locations typically due to the “collapse” of the pelvic floor, particularly due to weakness and/or tears/stretching of the pelvic floor muscles and supporting tissues. In women, there are generally three recognised types of POP; namely, anterior vaginal wall prolapse (which can be characterised by a protuberance of the bladder and/or urethra into the front wall of the vagina), posterior vaginal wall prolapse (which is characterised by a portion of the bowel or rectum protruding into the back wall of the vagina) and apical vaginal prolapse wherein the uterus, or the top part of vagina, may drop down into the vaginal canal. In any case, POP can cause a range of different symptoms including vaginal bulge, discomfort (e.g., a feeling of pressure or fullness), difficult bowel movements, and loss of bladder control (e.g., difficultly in urinating or, commonly, urinary leakage or incontinence).

In many cases, POP in women can be managed effectively by regular exercise (e.g., Kegel exercises to strengthen the pelvic floor muscles) and lifestyle choices or changes (e.g., the risk of developing POP is significantly increased by smoking and overweight; www.voicesforpfd.org), and/or the use of a pessary or vaginal support device. However, in more serious cases, surgical intervention may be recommended, particularly for women with more severe symptoms related to quality of life, bladder, bowel and sexual function. Numerous reconstructive and obliterative surgical approaches are available for POP, and until relatively recently, the use of synthetic mesh (i.e. polypropylene transvaginal mesh) was commonly employed, particularly for surgery for a cystocele (bladder protruding into the front wall of the vagina).

However, due to complications such as mesh erosion, dyspareunia, hispareunia, pelvic pain and increased re-operation rates and associated reduced quality of life, the use of synthetic mesh for POP surgery has been withdrawn in Australia and other countries. Consequently, gynaecological surgeons are returning to native tissue repair (NTR), however such techniques show a high failure rate (e.g., in one study investigating NTR for stage 2 anterior vaginal wall prolapse, it was found that 33% of patients required secondary prolapse compartment procedures from 0.6 to 13 years later; Lavelle R S et al.,195(4 Pt 1):1014-1020, 2016).

To this end, trials have been conducted using various biological grafts (e.g., human dermal cadaveric allografts and mammalian extracellular matrix (ECM) xenografts) to augment POP surgery, but it has been considered that there is insufficient evidence to support their use over standard NTR techniques (Advances in Female Pelvic Medicine and reconstructive Surgery, eds. HW Brown and RG Rogers, Elsevier, Philadelphia, PA, United States of America, 2021), and at this time, in Australia at least, such biological grafts are unavailable for POP surgery due to the lack of evidence of efficacy, and also as an aftermath of the abovementioned synthetic mesh complications (www.tga.gov.au/alert/tga-actions-after-review-urogyaecological-surgical-mesh-implants#actions).

Other surgical techniques using biological grafts or absorbable mesh to augment POP have been trialled, with systemic reviews based on low quality evidence demonstrating minimal advantage compared with NTR regarding rates on awareness of prolapse or reoperation (Maher C, Feiner B, Baessler K, Christmann-Schmid C, Haya N, Marjoribanks J. Transvaginal mesh or grafts compared with native tissue repair for vaginal prolapse. Cochrane Database Syst Rev. 2016; 2:CD012079). Low to moderate quality evidence suggests higher recurrence rates for anterior prolapse after NTR that with biological grafts.

Thus, there is an urgent need for the identification and development of novel non-mesh approaches for the treatment of POP such as techniques which may support and enhance pelvic floor native tissue repair as well as reduce the failure rate following surgery.

The present disclosure is based on products derived from whole blood which facilitate augmentation of native tissue repair and to provide tissue augmentation and physiological support. Disclosed herein are compositions (scaffolds) and methods for producing such compositions (scaffolds) which are particularly useful for providing mechanical support in pelvic organ prolapse.

In a first aspect, the present disclosure provides a method of producing a scaffold graft material or platelet rich plasma (PRP) graft for tissue repair and/or tissue support or reinforcement in a subject, comprising the steps of:

In one example, the method further comprises harvesting the pelleted graft material.

In a first embodiment, the method comprises producing a scaffold graft material, comprising the steps of:

In one example, the method further comprises:

In one example, the method further comprises (iv) harvesting the densely coagulated material.

In a second embodiment, the method comprises preparation of an autologous PRP graft material, the method comprising the steps of:

In one example, the coagulation activator is a gluconate salt. In one example, the gluconate salt is calcium gluconate.

In one example, the first separation force is between 3000-4000 RPM (900-1,600×g) for a first period of time of between 7-12 min.

In one example, the platelet-rich plasma (PRP) and gluconate salt are in a ratio of between 0.5:2-3:8 v/v.

In one example, whole blood is combined with a citrate salt prior to obtaining PRP. In another example, the citrate salt is sodium citrate.

In one example, the PRP concentration is at least 4-6 times the baseline concentration of platelets in the whole blood.

In one example, the second separation force is between 3,500-4,500 RPM (2000-3,200×g) for a second period of time of between 55-75 min.

In one example, the tissue repair and/or support is tissue repair and/or tissue support or reinforcement of a vaginal wall. In one example, the vaginal wall is the anterior vaginal wall. In another example the vaginal wall is the posterior vaginal wall.

The whole blood can comprise a blood sample from a single donor or from multiple donors mixed together to obtain a single blood sample. The blood sample can be obtained from the same subject who will receive the scaffold graft material. Thus, the blood is autologous to the recipient. The blood sample can also be obtained from a non-autologous subject or donor or multiple donors. Thus, the blood sample can be obtained from a heterologous subject or donor or multiple donors. In one example, the blood is collected into a suitable receptacle or container. In one example, the receptacle or container is glass. In another example, the receptacle or container is a borosilicate glass container. In one example the whole blood sample is from the said subject such that the method produces an autologous scaffold graft material for tissue repair and/or support in the subject. The blood sample may therefore be autologous or allogeneic to the subject.

The blood sample may be combined with one or more anti-coagulation agents and one or more coagulants. In a particular example the anti-coagulant and the coagulation activator are combined prior to the addition of whole blood. In one example, the anti-coagulation agent and the coagulation activator and blood sample are provided in sequential order. In one example, the blood sample is combined with the anti-coagulant prior to the addition of coagulant to avoid immediate clotting of the blood.

In one example, the densely coagulated material (i.e. scaffold graft material) according to the first embodiment comprises a substantially homogenous mixture of plasma, platelets, red blood cells and white blood cells. In one example, the densely coagulated material is substantially free of red blood cells and white blood cells. In one example, the proportion of red and white blood cells in the densely coagulated material comprises less than about 10%, preferably less than about 5% red blood cells and less than about 10%, preferably less than about 5% white blood cells. In one example, the densely coagulated material comprises substantially all of the platelets. In one example, the densely coagulated material comprises at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% of the platelets in the whole blood. In one example, the densely coagulated material comprises about 30% or less of the water content of plasma and plasma proteins and solids. In another example, the densely coagulated material comprises about 95-100% platelets, less than 10% red and white blood cells and about 30% water content of plasma.

In one example, the supernatant according to the first embodiment comprises about 70% plasma. In another example the supernatant comprises at least about 90% of the red blood cells and at least about 90% of the white blood cells in the whole blood.

In one example, the anti-coagulant agent is selected from heparin, ethylenediaminetetraacetic acid (EDTA), citrate, oxalate, thrombin inhibitors or other factor inhibitors. In another example, the anti-coagulation agent is sodium citrate. In one example, the sodium citrate is provided in solution in an amount of 2 ml per 18-20 ml of whole blood. In one example, the sodium citrate is provided as a solution of between about 1% and 5% (v/v), preferably about 3 to 4% (v/v), more preferably about 3.2% (v/v).

In one example, the coagulation activator initiates aggregation of fibrin present in the whole blood. In another example, the coagulation activator is a calcium salt, iron (ferrous) salt, aluminium salt, sodium salt or zinc salt. In another example, the coagulation factor is calcium chloride or calcium sulphate. In another example, the coagulation factor is calcium gluconate. In another example, the calcium gluconate is provided in an amount of 2 mL per 18-20 ml of whole blood. In another example, the calcium gluconate is provided as a solution of about 10%.

In one example according to the first embodiment, the volumes of the whole blood, anti-coagulant agent and coagulation activator in the scaffold graft material are in a ratio of about 8-12:1:1 v/v. In one example, the volumes of the whole blood, anti-coagulant agent and coagulation activator are in a ratio of about 9:1:1 v/v. In one example, the whole blood, sodium citrate, calcium gluconate are in a ratio of about 8-12:1:1 v/v, more preferably 9:1:1 v/v.

In one example according to the first embodiment, the anti-coagulant agent, coagulant activator and whole blood are exposed to a single separation force resulting in the densely coagulated material (i.e. scaffold graft material) and supernatant fractions. In a further example, the method comprises a single centrifugation step.

In one example according to the first embodiment, the densely coagulated material (i.e. scaffold graft material) is an homogenous mixture of platelets, red blood cells, white blood cells and plasma. In another example the densely coagulated material is not a multilayered structure. In another example the densely coagulated material is not a soft jelly-like material.

In one example, the method of the first embodiment does not require immediate clotting of the blood or the step of aggregating the fibrin prior to centrifugation.

The method of the first embodiment may also include modifying the densely coagulated material after harvest. In one example, the modification comprises washing or blotting the material on an absorbent material.

In a second aspect, the disclosure provides a scaffold graft material, produced by or obtainable by, the method of the first aspect. In one example, the scaffold material is an autologous scaffold graft material according to the first embodiment. In one example, the scaffold material is the autologous graft material according to the second embodiment.

In a third aspect, the disclosure provides a scaffold graft material, preferably an autologous scaffold material, for tissue repair and/or tissue support or reinforcement in a subject produced from whole blood, wherein the scaffold material is a flexibly solid non-gel homogenous material comprising about 95-100% platelets, less than 10% red and white blood cells and at least about 30% plasma. The platelets can further include inactivated and activated platelets. In a particular example, the scaffold graft comprises the densely coagulated material as described herein.

In a fourth aspect, the disclosure provides a scaffold graft material for tissue repair and/or tissue support or reinforcement to a pelvic organ in a female subject, wherein the scaffold material is a flexibly solid non-gel material comprising plasma, platelets and fibrin and being substantially free of red and white blood cells, and wherein the scaffold comprises an ultimate tensile strength of at least 3 MPa.

The scaffold graft material can comprise physical properties that allow it to be suturable but also of sufficient strength to provide biomechanical reinforcement to the site of prolapse, for example to the anterior and posterior vaginal walls. In one example, the scaffold material comprises an ultimate tensile strength of between 3 and 5 Mpa. In another example, the scaffold material comprises a suture retention strength of at least about 20N (Newton). In certain examples, the scaffold graft material forms a disc of relatively uniform cross-sectional thickness following centrifugation. In a further example, the diameter of the scaffold disc is in the range of about 3-5 cm, more preferably about 5 cm. In a further example, the thickness of the scaffold disc is about 1.5 to 3 mm). In some examples, the scaffold disc further comprises a surgical glue applied to one or both sides of the disc. In situ this may represent the anterior and/or posterior side. Suitable surgical glues will be familiar to persons skilled in the art. Examples include fibrin glue, albumin-glutaraldehyde or a cyanoacrylate-based tissue adhesive such as octyl-cyanoacrylate.

In a fifth aspect, the disclosure provides an autologous PRP graft material, the graft material comprising platelet rich plasma (PRP) and a gluconate salt, wherein the ratio of the gluconate salt to platelet-rich plasma is between 0.5:2-3:8 v/v. The PRP may be used for skin defects due to trauma, diabetes, or cancer, vascular injury, tendon injury, visceral injury, or any soft tissue damage requiring reinforcement or accelerating tissue reinforcement and augmentation.

In one example according to the fifth aspect, the gluconate salt is calcium gluconate. In one example, the ratio of the gluconate salt to platelet-rich plasma is 1:6 v/v. In another example, the ratio of the gluconate salt to platelet-rich plasma is 1:4 v/v. In a further example, the ratio of the gluconate salt to platelet-rich plasma is 2:6 v/v. In one example, the autologous graft material is a tissue reinforcement autologous graft material. In a further example, the autologous graft material is an autologous graft composite.

In one example, according to the fifth aspect, the autologous graft material (PRP graft) is enriched in platelets. In one example, the PRP graft is substantially free of red blood cells. In one example, the PRP is a jelly-like material.

The scaffold graft material described herein according to the fourth aspect or the autologous PRP graft material according to the fifth aspect, can further comprise optional components that can be added either during or after preparing the scaffold graft material. The scaffold graft material may include one or more materials to assist in strengthening the graft. In one example, the optional component is muscle, for example skeletal muscle. In one example, the graft further comprises a biodegradable scaffold material to further strengthen the graft.

For example, the biodegradable material may be a zinc, copper, magnesium or iron based alloy. In another example, the biodegradable material is a polymer (e.g. biopolymer). The polymer may be synthetic polymer, for example polyorthoester, polyphosphoester, polyanhydride, polyester-amide or polyamide. The polymer may be a natural polymer, for example chitosan, alginate, guar gum, starch, carrageenan, albumin or gelatin. In another example, the optional component is a growth factor. The growth factor may include one or more of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), transforming growth factor beta (TGF-β), and fibroblast growth factor (FGF).

In a sixth aspect, the disclosure provides a method of treating a subject afflicted with pelvic organ prolapse (POP), said method comprising the steps of:

In one example, the POP is anterior vaginal wall prolapse. In another example, the POP is uterine prolapse. In another example, the POP is selected from anterior compartment prolapse, posterior compartment prolapse, anterior and posterior prolapse, global prolapse, anterior and apical prolapse, posterior and apical prolapse and apical prolapse.

In some examples, the method of treatment includes applying one or more scaffold graft materials to the site depending on the extent of the injury. The scaffolds can be overlapping on placed side-by-side. In other examples, the method of treatment includes a further treatment subsequent to a failure of previous treatment.

In one example, the subject has one or more of the following age>35 years, body mass index>24, obesity, menopause, previous forcepts delivery, previous caesarean delivery, hysterectomy. In one example, the subject has a concurrent complaint. In another example, the concurrent complaint is selected from voiding dysfunction, recurrent urinary tract infection (UTI), bowel dysfunction, sexual dysfunction, lichen sclerosis, atrophic vaginitis and vulvodynia.

In one example, the method of the sixth aspect further comprises the step of surgical repair of the anterior and/or posterior wall fascia of the vagina. In one example, the surgical repair comprises suturing.

In some examples, one or more scaffold grafts are affixed to the site of surgical repair.